Brain dopamine neurons appear to be injured by chronic cocaine exposure in human users. Striatal VMAT2 binding sites, VMAT2 IR, and dopamine concentrations, as well as midbrain melanized neurons and DAT mRNA are decreased in post mortem human samples that we have examined. Cocaine-related neurotoxicity in humans could contribute to disordered reward experience, and specific symptoms such as craving and depression. Goal #1 is to confirm that dopamine neurons are lost in midbrain, paralleled by loss of striatal dopaminergic fibers, in human cocaine users. Specific Aim #1 is to test the hypotheses that dopamine neurons are lost in the anterior dorsal tier of pars compacta of human cocaine users. Dopamine cell numbers will be quantitated in major dopamine nuclei at four AP levels in available specimens, using stereological methods and naturally occurring melanin inclusions and tyrosine hydroxylase (TH) immunohistochemical staining to identify dopamine cells. GABA neurons will be evaluated to assess the specificity of the cocaine effect. VMAT2 decreases could result from cocaine-induced regulation of VMAT2 protein, or neurotransmitter vesicles. Goal #2 is to clarify if human cocaine users experience dopamine-specific regulation of neurotransmitter vesicle levels. Specific Aim #2 is to test the hypotheses that the vesicle-associated protein, synaptotagmin 1, decreases, while the synapse-associated protein, syntaxin, does not, specifically in dopamine neurons. VMAT2, syntaxin, and synaptotagmin 1 mRNA levels will be quantitated in midbrain regions, while further characterization of synaptotagmin subtypes and other synaptic protein levels will be assessed in striatum. Dopamine neurons have specific, topographically organized projections that subserve distinct motor versus limbic functions. Goal #3 is to better understand the anatomical extent that dopamine function is affected in cocaine users and to determine if regional pattems exist in stdatum. Distinct among dopamine markers, striatal dopamine transporter (DAT) function is increased, and could be related to the toxic effects of long-term cocaine exposure. Specific Aim #3 will test the hypothesis that cocaine users display both decreased striatal VMAT2 binding sites and increased DAT binding in a co-varying pattern that is most intense in anterior ventromedial striatum. DATNMAT2 expression in amygdalar nuclei subserving reward/emotional functions will also be examined. Methamphetamine toxicity in mice appears to involve excessive sodium influx into dopamine neurons, inducing increases in energetic (COX-l) and sodium exchange (NHE-1) enzymes, which were both increased in six cocaine users. Goal #4 is to confirm these increases and determine if induction of related genes occurs in human cocaine users. Specific Aim # 4 is to test the hypotheses that COX-1, NHE-1 and related mRNA's are increased in dopamine neurons.